This invention relates to antagonist monoclonal antibodies (mAb) that bind to the Tie2 receptor and to the use of such antibodies for therapeutic purposes.
Chronic proliferative diseases are often accompanied by profound angiogenesis, which can contribute to or maintain an inflammatory and/or proliferative state, or which leads to tissue destruction through the invasive proliferation of blood vessels. (Folkman, EXS 79:1-8, 1997; Folkman, Nature Medicine 1:27-31, 1995; Folkman and Shing, J. Biol. Chem. 267:16931, 1992).
Angiogenesis or neovascularisation is the process of development of new or replacement blood vessels. It is a necessary and normal process by which the vasculature is established in the embryo. Angiogenesis does not occur, in general, in most normal adult tissues, exceptions being sites of ovulation, menses and wound healing.
Many diseases, however, are characterized by persistent and unregulated angiogenesis. For instance, in arthritis, new capillary blood vessels invade the joint and destroy cartilage (Colville-Nash and Scott, Ann. Rheum. Dis., 51, 919,1992). In diabetes (and in many different eye diseases), new vessels invade the macula or retina or other ocular structures, and may cause blindness (Brooks et al., Cell, 79, 1157, 1994). The process of atherosclerosis has been linked to angiogenesis (Kahlon et al., Can. J. Cardiol. 8, 60, 1992). Tumor growth and metastasis have been found to be angiogenesis-dependent (Folkman, Cancer Biol, 3, 65, 1992; Denekamp, Br. J. Rad. 66, 181, 1993; Fidler and Ellis, Cell, 79, 185, 1994).
The recognition of the involvement of angiogenesis in major diseases has been accompanied by research to identify and develop angiogenesis inhibitors. Angiogenesis occurs in many stages and inhibitors are generally classified in response to discrete targets in the angiogenesis cascade, such as activation of endothelial cells by an angiogenic signal; synthesis and release of degradative enzymes; endothelial cell migration; proliferation of endothelial cells; and formation of capillary tubules. Literature reports indicate that inhibitors of angiogenesis, working by diverse mechanisms, are beneficial in diseases such as cancer and metastasis (O""Reilly et al., Cell, 79, 315, 1994; Ingber et al., Nature, 348, 555, 1990), ocular diseases (Friedlander et al., Science, 270, 1500, 1995), arthritis (Peacock et al., J. Exp. Med. 175, 1135, 1992; Peacock et al., Cell. Immun. 160, 178, 1995) and hemangioma (Taraboletti et al., J. Natl. Cancer Inst. 87, 293, 1995).
In recent years, it has become clear that while angiogenesis is a complex, multicellular phenomena, specific ligands and their receptors play a key role. In particular, a combination of studies suggest that the Tie2 receptor and its ligand are important in angiogenesis.
Tie2 receptor has been located in endothelial cells of all forming blood vessels and in the endocardium of mouse embryos (Korhonen et al., Blood 80:2548-2555, 1992). Selective patterns of expression in endothelial cells during embryonic development has also been demonstrated (Schlaeger et al., Development 121:1089-1098, 1995).
In adult tissues, Tie mRNA cannot be observed in skin, except at sites of active wound healing where the proliferating capillaries in the granulation tissue contain abundant Tie mRNA (Korhonen et al., Blood 80:2548-2555, 1992). Further, Tie receptor is expressed in the vascular endothelium of metastasizing melanomas (Kaipainen et al., Cancer Res. 54:6571-6577, 1994). While Tie receptor expression is down-regulated in the established vasculature, it is up-regulated in angiogenesis that occurs in the ovary during ovulation, in wounds and in tumor (breast, melanoma and renal cell carcinoma) vasculature, consistent with prevailing views that angiogenesis in the adult borrows from embryonic angiogenic mechanisms.
Homozygous mice with a Tie2 knockout, or carrying a transgene encoding a xe2x80x9cdominant-negativexe2x80x9d Tie2 receptor, confirmed that the Tie2 receptor is critical for embryonic development (Dumont et al., Genes Dev. 8:1897-1909, 1994; Sato et al., Nature 376:70-74, 1995). Embryonic death in these mice occurred due to vascular insufficiency and there were dramatically reduced numbers of endothelial cells. Vasculogenesisxe2x80x94that is the differentiation of endothelial cells and the in situ formation of vesselsxe2x80x94appeared relatively normal in mice lacking Tie2. The subsequent sprouting and remodeling resulting in formation of vessel branches (angiogenesis) was drastically reduced in the Tie2 mutant mice embryos. This lack of sprouting and angiogenesis resulted in substantial growth retardation, particularly of the brain, neural tube and heart, resulting in lack of viability. These results exemplify the critical importance of Tie2 in angiogenesis. This is significant, as angiogenesis is regulated by a number of growth factors. Interestingly, Flk1 (VEGF receptor) knockout mice exhibit embryo lethal defects in vasculogenesis, that occur earlier than those of Tie2 disruption. Disruption of the Tie1 receptor yields a much different, and later, defective phenotype; the mouse embryo dies late in development due to hemorrhage resulting from defective integrity of an otherwise well-formed vasculature. Taken together, these studies suggest that the VEGF/Flk1 and Tie systems operate in sequential fashion, with Tie2 having a critical role in angiogenesis.
Recently, two ligands for the Tie2 receptor have been reported. Angiopoietin-1 binds and induces the tyrosine phosphorylation of Tie2 and its expression in vivo is in close proximity with developing blood vessels (Davis et al., Cell 87:1161-1169,1996). Mice engineered to lack Angiopoietin-1 display angiogenic deficits reminiscent of those previously seen in mice lacking Tie2 receptors, demonstrating that Angiopoietin-1 is a primary physiologic ligand for Tie2 and that Tie2 has critical in vivo angiogenic actions (Suri et al., Cell 87:1171-1180, 1996). Angiopoietin-2 was identified by homology screening and shown to be a naturally occurring antagonist for Tie2 receptors. Transgenic overexpression of Angiopoietin-2 disrupts blood vessel formation in the mouse embryo (Maisonpierre et al., Science 277:55-60, 1997). Together, these results support a role for Tie2 receptors in angiogenesis.
The Tie1 and Tie2 receptors are single-transmembrane, tyrosine kinase receptors (Tie stands for Tyosine kinase receptors with immunoglobulin and EGF homology domains). They are the only receptor tyrosine kinases, other than those receptors for VEGF, that are largely restricted to endothelial cells in their expression. Both have been cloned and reported by several groups (Dumont et al., Oncogene 8:1293-1301, 1993; Partanen et al., Mol. Cell Biol. 12:1698-1707, 1992; Sato et al., Proc. Natl. Acad. Sci. USA 90:9355-9358, 1993).
The Tie receptors are proteins of approximately 125 kDa, with a single putative transmembrane region. The extracellular domain of these receptors is uniquely divided into three regions that have a pattern of cysteine expression found in EGF-like domains; two regions that have some weak homology to and structural characteristics of immunoglobulin-like domains; and three regions with homology to the fibronectin III repeat structure. The intracellular portion of Tie2 is most closely related (xcx9c40% identity) to the kinase domains of FGF-R1, PDGF-R and c-kit. The intracellular portions of Tie2 contain all of the features of tyrosine kinases, including a GXGXXG ATP binding site consensus sequence and typical tyrosine kinase motifs (i.e., HRDLAARN and DFGL).
Based upon the importance of Tie2 receptors in angiogenesis, inhibition of Tie2 activity is predicted to interrupt angiogenesis, providing disease-specific therapeutic effects. Recently, Lin et al. (J. Clin. Invest. 100:2072-2078, 1997) has shown that exogenously administered soluble Tie2 receptor inhibited angiogenesis and cancer growth in animal models. Clearly, there is a need to develop high affinity, potent antagonist antibodies to the Tie2 receptor which will have sufficient activity to work in vivo at therapeutically acceptable concentrations.
Tie2 receptors are expressed on hematopoietic progenitors, including CD34+ cells, megakaryocyte progenitors and megakaryocyte-derived cell lines (Kukk et al., Brit J Haematol. 98:195-203, 1997; Batard et al., Blood 87:2212-2220, 1996). The Tie2 receptor has homology to other hematopoietic growth factor receptors such as c-kit (Chabot et al, Nature 335:88, 1988; Yarden et al, EMBO J 6:3341, 1987) and flk-2 (Matthews et al, Cell 65:1143, 1991). The expression of Tie2 decreased on more mature hematopoietic cells, although it was maintained on a significant fraction of cells during megakaryocytic differentiation (Batard). These data, taken together, suggest that Tie2 is a receptor for a hematopoietic growth factor acting on early progenitors and during differentiation of the megakaryocytic lineage.
One aspect of the present invention is a Tie2 receptor antagonist antibody having the identifying characteristics of monoclonal antibody 12H8 or 6A3.
Another aspect of the invention is an antibody comprising a heavy chain variable region polypeptide as set forth in SEQ ID NO: 2 and a light chain variable region polypeptide as set forth in SEQ ID NO: 4 and isolated polynucleotides encoding them.
Another aspect of the invention is a heavy chain CDR having an amino acid sequence as set forth in SEQ ID NO: 5, 6 or 7.
Another aspect of the invention is a light chain CDR having an amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.
Another aspect of the invention is a hybridoma having the identifying characteristics of cell line 12H8 or 6A3.
Another aspect of the invention is a method for inhibiting angiogenesis in a animal comprising administering an effective dose of an Tie2 receptor antagonist antibody having the identifying characteristics of monoclonal antibody 12H8 or 6A3.
Another aspect of the invention is a method for expansion of marrow cells transfected with a gene for gene therapy comprising treating the marrow cells with a Tie2 receptor antagonist antibody.
Another aspect of the invention is a method for expansion of cord blood, marrow or peripheral blood cells for transplant comprising treating the cells with a Tie2 receptor antagonist antibody.
Another aspect of the invention is a method to enhance the survival of hematopoietic progenitor cells in a mammal comprising treating the cells with a Tie2 receptor antagonist antibody.
Another aspect of the invention is a method to increase the proliferation of megakaryocyte cells or megakaryotcyte progenitor cells to increase platelet numbers in a mammal comprising treating the cells with a Tie2 receptor antagonist antibody.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.
As used herein, the term xe2x80x9cinhibiting angiogenesisxe2x80x9d means decreasing the development of new or replacement blood vessels (neovascularization).
As used herein, the term xe2x80x9cantagonist activityxe2x80x9d refers to the activity of an antibody that binds to Tie2 receptor and inhibits angiogenesis.
As used herein, the term xe2x80x9ctreatingxe2x80x9d and derivatives thereof means prophylactic, palliative or therapeutic therapy.
The present invention provides a variety of antibodies, including altered antibodies and fragments thereof directed against Tie2 receptor, which are characterized by antagonist activity. Exemplary Tie2 receptor antagonist antibodies are the murine monoclonal antibodies 12H8 or 6A3.
xe2x80x9cAntibodiesxe2x80x9d refers to immunoglobulins which can be prepared by conventional hybridoma techniques, phage display combinatorial libraries, immunoglobulin chain shuffling and humanization techniques. Also included are fully human monoclonal antibodies. As used herein, xe2x80x9cantibodyxe2x80x9d also includes xe2x80x9caltered antibodyxe2x80x9d which refers to a protein encoded by an altered immunoglobulin coding region, which may be obtained by expression in a selected host cell. Such altered antibodies are engineered antibodies (e.g., chimeric or humanized antibodies) or antibody fragments lacking all or part of an immunoglobulin constant region, e.g., Fv, Fab, Fabxe2x80x2 or F(ab)2 and the like. These antibody products are useful in therapeutic and pharmaceutical compositions for treating angiogenic and proliferative (including inflammatory) diseases and other conditions which are caused by excessive or inappropriate angiogenesis. Exemplary angiogenic diseases include diabetic retinopathy and macular degeneration. Exemplary proliferative diseases include cancer, arthritis, psoriasis and atherosclerosis. Alternatively, the antibodies of the invention can be coupled to toxins, antiproliferative drugs or radionuclides to kill cells in areas of excessive Tie2 receptor expression and/or angiogenesis.
xe2x80x9cAltered immunoglobulin coding regionxe2x80x9d refers to a nucleic acid sequence encoding an altered antibody of the invention. When the altered antibody is a complementarity determining region-grafted (CDR-grafted) or humanized antibody, the sequences that encode the CDRs from a non-human immunoglobulin are inserted into a first immunoglobulin partner comprising human variable framework sequences. Optionally, the first immunoglobulin partner is operatively linked to a second immunoglobulin partner.
xe2x80x9cFirst immunoglobulin partnerxe2x80x9d refers to a nucleic acid sequence encoding a human framework or human immunoglobulin variable region in which the native (or naturally-occurring) CDR-encoding regions are replaced by the CDR-encoding regions of a donor antibody. The human variable region can be an immunoglobulin heavy chain, a light chain (or both chains), an analog or functional fragments thereof. Such CDR regions, located within the variable region of antibodies (immunoglobulins) can be determined by known methods in the art. For example Kabat et al. in xe2x80x9cSequences of Proteins of Immunological Interestxe2x80x9d, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987) disclose rules for locating CDRs. In addition, computer programs are known which are useful for identifying CDR regions/structures.
xe2x80x9cSecond immunoglobulin partnerxe2x80x9d refers to another nucleotide sequence encoding a protein or peptide to which the first immunoglobulin partner is fused in frame or by means of an optional conventional linker sequence (i.e., operatively linked). Preferably, it is an immunoglobulin gene. The second immunoglobulin partner may include a nucleic acid sequence encoding the entire constant region for the same (i.e., homologous, where the first and second altered antibodies are derived from the same source) or an additional (i.e., heterologous) antibody of interest. It may be an immunoglobulin heavy chain or light chain (or both chains as part of a single polypeptide). The second immunoglobulin partner is not limited to a particular immunoglobulin class or isotype. In addition, the second immunoglobulin partner may comprise part of an immunoglobulin constant region, such as found in a Fab, or F(ab)2 (i.e., a discrete part of an appropriate human constant region or framework region). Such second immunoglobulin partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, e.g., as part of a phage display library, or a sequence encoding a protein for analytical or diagnostic detection, e.g., horseradish peroxidase, xcex2-galactosidase, etc.
The terms Fv, Fc, Fd, Fab, Fabxe2x80x2 or F(abxe2x80x2)2 are used with their standard meanings. See, e.g., Harlow et al. in xe2x80x9cAntibodies A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, (1988).
As used herein, an xe2x80x9cengineered antibodyxe2x80x9d describes a type of altered antibody, i.e., a full-length synthetic antibody (e.g., a chimeric or humanized antibody as opposed to an antibody fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope. For example, such molecules may include antibodies characterized by a humanized heavy chain associated with an unmodified light chain (or chimeric light chain), or vice versa. Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity. These antibodies can comprise replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein.
A xe2x80x9cchimeric antibodyxe2x80x9d refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.
A xe2x80x9chumanized antibodyxe2x80x9d refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins. In addition, framework support residues may be altered to preserve binding affinity. See, e.g., Queen et al., Proc. Natl Acad Sci USA, 86, 10029-10032 (1989), Hodgson et al., Bio/Technoloy 9, 421 (1991). Furthermore, as decribed herein, additional residues may be altered to preserve the antagonist activity of the donor antibody.
The term xe2x80x9cdonor antibodyxe2x80x9d refers to a monoclonal or recombinant antibody which contributes the nucleic acid sequences of its variable regions, CDRs or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody. Donor antibodies suitable for use in this invention is a murine antagonist monoclonal antibody designated as 12H8 and 6A3.
The term xe2x80x9cacceptor antibodyxe2x80x9d refers to monoclonal or recombinant antibodies heterologous to the donor antibody, which contributes all, or a portion, of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions or V region subfamily consensus sequences to the first immunoglobulin partner. Preferably, a human antibody is the acceptor antibody.
xe2x80x9cCDRsxe2x80x9d are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs or CDR regions in the variable portion of an immunoglobulin. Thus, xe2x80x9cCDRsxe2x80x9d as used herein refers to all three heavy chain CDRs, or all three light chain CDRs or both all heavy and all light chain CDRs, if appropriate.
CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs share or retain the same antigen binding specificity and/or antagonist ability as the donor antibody from which they were derived, yet may exhibit increased affinity for the antigen. An exemplary process for obtaining analogs is affinity maturation by means of phage display technology as reviewed by Hoogenboom, Trends in Biotechnology 15, 62-70 (1997); Barbas et al., Trends in Biotechnology 14, 230-234 (1996); and Winter et al., Ann. Rev. Immunol. 12, 433-455 (1994) and described by Irving et al., Immunotechnology 2, 127-143 (1996).
By xe2x80x9csharing the antigen binding specificity or antagonist abilityxe2x80x9d is meant, for example, that although mAb 12H8 or 6A3 may be characterized by a certain level of antagonist activity, a CDR encoded by a nucleic acid sequence of 12H8 or 6A3 in an appropriate structural environment may have a lower or higher activity. It is expected that CDRs of 12H8 or 6A3 in such environments will nevertheless recognize the same epitope(s) as 12H8 or 6A3.
A xe2x80x9cfunctional fragmentxe2x80x9d is a partial heavy or light chain variable sequence (e.g., minor deletions at the amino or carboxy terminus of the immunoglobulin variable region) which retains the same antigen binding specificity and/or antagonist ability as the antibody from which the fragment was derived.
An xe2x80x9canalogxe2x80x9d is an amino acid sequence modified by at least one amino acid, wherein said modification can be chemical or a substitution or a rearrangement of a few amino acids (i.e., no more than 10) and corresponding nucleic acid sequences, which modification permits the amino acid sequence to retain the biological characteristics, e.g., antigen specificity and high affinity, of the unmodified sequence. Exemplary nucleic acid analogs include silent mutations which can be constructed, via substitutions, to create certain endonuclease restriction sites within or surrounding CDR-encoding regions.
Analogs may also arise as allelic variations. An xe2x80x9callelic variation or modificationxe2x80x9d is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to degeneracy in the genetic code or may be deliberately engineered to provide desired characteristics. These variations or modifications may or may not result in alterations in any encoded amino acid sequence.
The term xe2x80x9ceffector agentsxe2x80x9d refers to non-protein carrier molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments of the donor antibody may be associated by conventional means. Such non-protein carriers can include conventional carriers used in the diagnostic field, e.g., polystyrene or other plastic beads, polysaccharides, e.g., as used in the BIAcore (Pharmacia) system, or other non-protein substances useful in the medical field and safe for administration to humans and animals. Other effector agents may include a macrocycle, for chelating a heavy metal atom or radioisotopes. Such effector agents may also be useful to increase the half-life of the altered antibodies, e.g., polyethylene glycol.
For use in constructing the antibodies, altered antibodies and fragments of this invention, a non-human species such as bovine, ovine, monkey, chicken, rodent (e.g., murine and rat) may be employed to generate a desirable immunoglobulin upon presentment with human Tie2 receptor or a peptide epitope therefrom. Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human mAb to the Tie2 receptor. Such hybridomas are then screened for binding and antagonist activity as described in the Examples section. Alternatively, fully human mAbs can be generated by techniques known to those skilled in the art and used in this invention.
Exemplary antagonist mAbs of the present invention is mAb 12H8 and 6A3, murine antibodies which can be used for the development of a chimeric or humanized molecule. The 12H8 and 6A3 mAbs are characterized by antagonist activity on human Tie2 receptor and subsequent blocking of the receptor""s signal transduction resulting in inhibition of angiogenesis. These mabs are produced by the hybridoma cell lines 12H8 and 6A3, respectively.
The present invention also includes the use of Fab fragments or F(abxe2x80x2)2 fragments derived from mAbs directed against Tie2 receptor as bivalent fragments. These fragments are useful as agents having antagonist activity at the Tie2 receptor. A Fab fragment contains the entire light chain and amino terminal portion of the heavy chain. An F(abxe2x80x2)2 fragment is the fragment formed by two Fab fragments bound by disulfide bonds. The mAbs 12H8 and 6A3 and other similar high affinity antibodies provide sources of Fab fragments and F(abxe2x80x2)2 fragments which can be obtained by conventional means, e.g., cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin, or by recombinant methods. These Fab and F(abxe2x80x2)2 fragments are useful themselves as therapeutic, prophylactic or diagnostic agents, and as donors of sequences including the variable regions and CDR sequences useful in the formation of recombinant or humanized antibodies as described herein.
The Fab and F(abxe2x80x2)2 fragments can be constructed via a combinatorial phage library (see, e.g., Winter et al., Ann. Rev. Immunol., 12:433-455 (1994)) or via immunoglobulin chain shuffling (see, e.g., Marks et al., Bio/Technology, 10:779-783 (1992)), wherein the Fd or VH immunoglobulin from a selected antibody (e.g., 12H8 or 6A3) is allowed to associate with a repertoire of light chain immunoglobulins, VL (or vK), to form novel Fabs. Conversely, the light chain immunoglobulin from a selected antibody may be allowed to associate with a repertoire of heavy chain immunoglobulins, vH (or Fd), to form novel Fabs. Tie2 receptor antagonist Fabs can be obtained by allowing the Fd of mAb 12H8 or 6A3 to associate with a repertoire of light chain immunoglobulins. Hence, one is able to recover neutralizing Fabs with unique sequences (nucleotide and amino acid) from the chain shuffling technique.
The mAb 12H8 or 6A3 may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various altered antibodies which are characterized by the antigen binding specificity of the donor antibody.
The nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified or fusion nucleic acid sequence into a plasmid for expression. For example, silent substitutions in the nucleotide sequence of the framework and CDR-encoding regions can be used to create restriction enzyme sites which facilitate insertion of mutagenized CDR and/or framework regions. These CDR-encoding regions can be used in the construction of the humanized antibodies of the invention.
The nucleic and amino acid sequences of the 12H8 heavy chain variable region is listed in SEQ ID NO: 1. The CDR amino acid sequences from this region are listed in SEQ ID Nos: 5, 6 and 7.
The nucleic and amino acid sequences of the 12H8 light chain variable region listed in SEQ ID NO: 3. The CDR amino acid sequences from this region are listed in SEQ ID Nos: 8, 9 and 10.
Taking into account the degeneracy of the genetic code, various coding sequences may be constructed which encode the variable heavy and light chain amino acid sequences and CDR sequences of the invention as well as functional fragments and analogs thereof which share the antigen specificity of the donor antibody. The isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs can be used to produce altered antibodies, e.g., chimeric or humanized antibodies or other engineered antibodies of this invention when operatively combined with a second immunoglobulin partner.
It should be noted that in addition to isolated nucleic acid sequences encoding portions of the altered antibody and antibodies described herein, other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions. Useful DNA sequences include those sequences which hybridize under stringent hybridization conditions to the DNA sequences. See, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982), pp. 387-389. An example of one such stringent hybridization condition is hybridization at 4xc3x97SSC at 65xc2x0 C., followed by a washing in 0.1xc3x97SSC at 65xc2x0 C. for one hour. Alternatively, an exemplary stringent hybridization condition is 50% formamide, 4xc3x97SSC at 42xc2x0 C. Preferably, these hybridizing DNA sequences are at least about 18 nucleotides in length, i.e., about the size of a CDR.
Altered immunoglobulin molecules can encode altered antibodies which include engineered antibodies such as chimeric antibodies and humanized antibodies. A desired altered immunoglobulin coding region contains CDR-encoding regions that encode peptides having the antigen specificity of an Tie2 receptor antibody, preferably a high-affinity antagonist antibody such as provided by the present invention, inserted into a first immunoglobulin partner such as a human framework or human immunoglobulin variable region.
Preferably, the first immunoglobulin partner is operatively linked to a second immunoglobulin partner. The second immunoglobulin partner is defined above, and may include a sequence encoding a second antibody region of interest, for example an Fc region. Second immunoglobulin partners may also include sequences encoding another immunoglobulin to which the light or heavy chain constant region is fused in frame or by means of a linker sequence. Engineered antibodies directed against functional fragments or analogs of the Tie2 receptor may be designed to elicit enhanced binding with the same antibody.
The second immunoglobulin partner may also be associated with effector agents as defined above, including non-protein carrier molecules, to which the second immunoglobulin partner may be operatively linked by conventional means.
Fusion or linkage between the second immunoglobulin partners, e.g., antibody sequences, and the effector agent may be by any suitable means, e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde and the like. Such techniques are known in the art and are described in conventional chemistry and biochemistry texts.
Additionally, conventional linker sequences which simply provide for a desired amount of space between the second immunoglobulin partner and the effector agent may also be constructed into the altered immunoglobulin coding region. The design of such linkers is well known to those of skill in the art.
In addition, signal sequences for the molecules of the invention may be modified by techniques known to those skilled in the art to enhance expression.
A preferred altered antibody contains a variable heavy and/or light chain peptide or protein sequence having the antigen specificity of mAb 12H8 or 6A3, e.g., the VH and VL chains. Still another desirable altered antibody of this invention is characterized by the amino acid sequence containing at least one, and preferably all of the CDRs of the variable region of the heavy and/or light chains of the murine antibody molecule 12H8 or 6A3 with the remaining sequences being derived from a human source, or a functional fragment or analog thereof.
In a further embodiment, the altered antibody of the invention may have attached to it an additional agent. For example, recombinant DNA technology may be used to produce an altered antibody of the invention in which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule, i.e., a polypeptide effector or reporter molecule. Other additional agents include toxins, antiproliferative drugs and radionuclides.
The second immunoglobulin partner may also be operatively linked to a non-immunoglobulin peptide, protein or fragment thereof heterologous to the CDR-containing sequence having antigen specificity to the Tie2 receptor. The resulting protein may exhibit both antigen specificity and characteristics of the non-immunoglobulin upon expression. That fusion partner characteristic may be, e.g., a functional characteristic such as another binding or receptor domain or a therapeutic characteristic if the fusion partner is itself a therapeutic protein or additional antigenic characteristics.
Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains or any discrete fragment thereof, such as the Fab or F(abxe2x80x2)2 fragments, a heavy chain dimer or any minimal recombinant fragments thereof such as an Fv or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor mAb, e.g., the 12H8 or 6A3 mAb. Such protein may be used in the form of an altered antibody or may be used in its unfused form.
Whenever the second immunoglobulin partner is derived from an antibody different from the donor antibody, e.g., any isotype or class of immunoglobulin framework or constant regions, an engineered antibody results. Engineered antibodies can comprise immunoglobulin constant regions and variable framework regions from one source, e.g., the acceptor antibody, and one or more (preferably all) CDRs from the donor antibody, e.g., the 12H8 or 6A3 mAb. In addition, alterations, e.g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region at the nucleic acid or amino acid levels, or the donor CDR regions may be made in order to retain donor antibody antigen binding specificity.
Such engineered antibodies are designed to employ one (or both) of the variable heavy and/or light chains of the Tie2 receptor mAb (optionally modified as described) or one or more of the heavy or light chain CDRs. The engineered antibodies of the invention exhibit antagonist activity.
Such engineered antibodies may include a humanized antibody containing the framework regions of a selected human immunoglobulin or subtype or a chimeric antibody containing the human heavy and light chain constant regions fused to the Tie2 receptor mAb functional fragments. A suitable human (or other animal) acceptor antibody may be one selected from a conventional database, e.g., the KABAT(copyright) database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the V region frameworks of the donor antibody or V region subfamily consensus sequences (on an amino acid basis) may be suitable to provide a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
Preferably, the heterologous framework and constant regions are selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA, and IgE. IgG1, k and IgG4, k are preferred. Particularly preferred is IgG 4, k. Most particularly preferred is the IgG4 subtype variant containing the mutations S228P and L235E (PE mutation) in the heavy chain constant region which results in reduced effector function. This IgG4 subtype variant is known herein as IgG4PE. See U.S. Pat. Nos. 5, 624,821 and 5,648,260.
The acceptor antibody need not comprise only human immunoglobulin protein sequences. For instance, a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding a non-immunoglobulin amino acid sequence such as a polypeptide effector or reporter molecule.
A particularly preferred humanized antibody contains CDRs of 12H8 or 6A3 mAb inserted onto the framework regions of a selected human antibody sequence. For antagonist humanized antibodies, one, two or preferably three CDRs from the 12H8 or 6A3 antibody heavy chain and/or light chain variable regions are inserted into the framework regions of the selected human antibody sequence, replacing the native CDRs of the human antibody.
Preferably, in a humanized antibody, the variable domains in both human heavy and light chains have been engineered by one or more CDR replacements. It is possible to use all six CDRs, or various combinations of less than the six CDRs. Preferably all six CDRs are replaced. It is possible to replace the CDRs only in the human heavy chain, using as light chain the unmodified light chain from the human acceptor antibody. Still alternatively, a compatible light chain may be selected from another human antibody by recourse to conventional antibody databases. The remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin.
The engineered humanized antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e.g., treatment of angiogenic diseases such as diabetic retinopathy and macular degeneration or treatment of proliferative diseases, such as cancer, arthritis, psoriasis and atherosclerosis.
It will be understood by those skilled in the art that an engineered antibody may be further modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (i.e., an analog). It is anticipated that heavy and light chain amino acids may be substituted by other amino acids either in the variable domain frameworks or CDRs or both. These substitutions could be supplied by the donor antibody or consensus sequences from a particular subgroup.
In addition, the constant region may be altered to enhance or decrease selective properties of the molecules of this invention. For example, dimerization, binding to Fc receptors, or the ability to bind and activate complement (see, e.g., Angal et al., Mol. Immunol, 30, 105-108 (1993), Xu et al., J. Biol. Chem, 269, 3469-3474 (1994), Winter et al., EP 307434-B).
An altered antibody which is a chimeric antibody differs from the humanized antibodies described above by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions, in association with human immunoglobulin constant regions for both chains. It is anticipated that chimeric antibodies which retain additional non-human sequence relative to humanized antibodies of this invention may elicit a significant erythropoietic response in humans. Such antibodies are useful in the prevention of and for treating angiogenic diseases such as diabetic retinopathy and macular degeneration or treatment of proliferative diseases, such as cancer, arthritis, psoriasis and atherosclerosis.
Preferably, the variable light and/or heavy chain sequences and the CDRs of mAb 12H8 or 6A3 or other suitable donor mAbs and their encoding nucleic acid sequences, are utilized in the construction of altered antibodies, preferably humanized antibodies, of this invention, by the following process. The same or similar techniques may also be employed to generate other embodiments of this invention.
A hybridoma producing a selected donor mAb, e.g., the murine antibody 12H8 or 6A3, is conventionally cloned and the DNA of its heavy and light chain variable regions obtained by techniques known to one of skill in the art, e.g., the techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989). The variable heavy and light regions containing at least the CDR-encoding regions and those portions of the acceptor mAb light and/or heavy variable domain framework regions required in order to retain donor mAb binding specificity, as well as the remaining immunoglobulin-derived parts of the antibody chain derived from a human immunoglobulin, are obtained using polynucleotide primers and reverse transcriptase. The CDR-encoding regions are identified using a known database and by comparison to other antibodies.
A mouse/human chimeric antibody may then be prepared and assayed for binding ability. Such a chimeric antibody contains the entire non-human donor antibody VH and VL regions, in association with human Ig constant regions for both chains.
Homologous framework regions of a heavy chain variable region from a human antibody are identified using computerized databases, e.g., KABAT(copyright), and a human antibody characterized by a homology to the V region frameworks of the donor antibody or V region subfamily consensus sequences (on an amino acid basis) to 12H8 or 6A3 is selected as the acceptor antibody. The sequences of synthetic heavy chain variable regions containing the CDR-encoding regions within the human antibody frameworks are designed with optional nucleotide replacements in the framework regions to incorporate restriction sites. This designed sequence is then synthesized using long synthetic oligomers. Alternatively, the designed sequence can be synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (PCR), and corrected for errors. A suitable light chain variable framework region can be designed in a similar manner.
A humanized antibody may be derived from the chimeric antibody, or preferably, made synthetically by inserting the donor mAb CDR-encoding regions from the heavy and light chains appropriately within the selected heavy and light chain framework. Alternatively, a humanized antibody of the invention may be prepared using standard mutagenesis techniques. Thus, the resulting humanized antibody contains human framework regions and donor mAb CDR-encoding regions. There may be subsequent manipulation of framework residues. The resulting humanized antibody can be expressed in recombinant host cells, e.g., COS, CHO or myeloma cells.
A conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the altered antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV or Rous Sarcoma virus promoter, and signal sequences, which can be derived from other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain. Preferably, this second expression vector is identical to the first except with respect to the coding sequences and selectable markers, in order to ensure, as much as possible, that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the altered antibody may reside on a single vector.
A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention. The humanized antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by an appropriate assay such as ELISA or RIA. Similar conventional techniques may be employed to construct other altered antibodies and molecules of this invention.
Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the pUC series of cloning vectors, such as pUC19, which is commercially available from supply houses, such as Amersham or Pharmacia, may be used. Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance) and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.
Similarly, the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vector. The vectors also contain selected regulatory sequences (such as CMV or Rous Sarcoma virus promoters) which direct the replication and expression of heterologous DNA sequences in selected host cells. These vectors contain the above-described DNA sequences which code for the engineered antibody or altered immunoglobulin coding region. In addition, the vectors may incorporate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.
The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art.
The components of such vectors, e.g., replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast and fungal expression may also be selected for this purpose.
The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobulin molecules thereof. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention.
Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably mammalian cells such as CHO, COS, a fibroblast cell (e.g., 3T3) and myeloid cells, and more preferably a CHO or a myeloid cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., supra.
Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs of the present invention (see, e.g., Plxc3xcckthun, A., Immunol. Rev., 130, 151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host. For example, various strains of E. coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Streptomyces, other bacilli and the like may also be employed.
Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera, and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8, 277-298, Plenum Press (1986) and references cited therein.
The general methods by which the vectors of the invention may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the altered antibody of the invention from such host cell are all conventional techniques. Likewise, once produced, the altered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention.
Yet another method of expression of the humanized antibodies may utilize expression in a transgenic animal, such as described in U.S. Pat. No. 4,873,316. This relates to an expression system using the animal""s casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.
Once expressed by the desired method, the engineered antibody is then examined for in vitro activity by use of an appropriate assay. Presently, surface plasmon resonance is employed to assess qualitative and quantitative binding of the engineered antibody to Tie2 receptor. Additionally, other in vitro assays such as the Matrigel vascularization model may also be used to determine antagonist activity prior to subsequent human clinical studies performed to evaluate the persistence of the engineered antibody in the body despite the usual clearance mechanisms.
Following the procedures described for humanized antibodies prepared from 12H8 or 6A3, one of skill in the art may also construct humanized antibodies from other donor antibodies, variable region sequences and CDR peptides described herein. Engineered antibodies can be produced with variable region frameworks potentially recognized as xe2x80x9cselfxe2x80x9d by recipients of the engineered antibody. Modifications to the variable region frameworks can be implemented to effect increases in antigen binding and antagonist activity without appreciable increased immunogenicity for the recipient. Such engineered antibodies may effectively treat a human for ischemic diseases such as myocardial infarction or cerebral stroke or treatment of vascular insufficiency diseases, such as diabetes. Such antibodies may also be useful in the diagnosis of those conditions.
This invention also relates to a method for enhancing angiogenesis in an mammal, particularly a human, which comprises administering an effective dose of an Tie2 receptor monoclonal antibody having antagonist activity. The mAb can include one or more of the antibodies or altered antibodies described herein or fragments thereof.
The therapeutic response induced by the use of the molecules of this invention is produced by the binding to the Tie2 receptor and the subsequent antagonist activity of the angiogenic process. Thus, the molecules of the present invention, when in preparations and formulations appropriate for therapeutic use, are highly desirable for persons susceptible to or experiencing angiogenic diseases such as diabetic retinopathy or macular degeneration or proliferative diseases, such as cancer, arthritis, psoriasis or atherosclerosis.
This invention also relates to a method for expansion of marrow cells transfected with a gene for gene therapy comprising treating the marrow cells with a Tie2 receptor antagonist antibody. Preferably, the expansion is ex vivo.
This invention also relates to a method for expansion of cord blood, marrow or peripheral blood cells for transplant comprising treating the cells with a Tie2 receptor antagonist antibody. Preferably, the expansion is ex vivo.
This invention also relates to a method to enhance the survival of hematopoietic progenitor cells in a mammal comprising treating the cells with a Tie2 receptor antagonist antibody.
This invention also relates to a method to increase the proliferation of megakaryocyte cells or megakaryotcyte progenitor cells to increase platelet numbers in a mammal comprising treating the cells with a Tie2 receptor antagonist antibody.
The mAb used in the methods of the invention can include one or more of the antibodies or altered antibodies described herein or fragments thereof. Preferably, the Tie2 receptor antagonist antibody used in the methods of the invention has the identifying characteristics of mAb 12H8 or 6A3.
The altered antibodies, antibodies and fragments thereof of this invention may also be used in conjunction with other antibodies, particularly human mAbs reactive with other markers (epitopes) responsible for the condition against which the engineered antibody of the invention is directed.
The Tie2 receptor antagonist antibodies of the invention can be formulated into pharmaceutical compositions and administered in the same manner as described for mature proteins. See, e.g., International Patent Application, Publication No. WO90/02762 (Mar. 22 1990). Generally, these compositions contain a therapeutically effective amount of an antagonist antibody of this invention and an acceptable pharmaceutical carrier. Suitable carriers are well known to those of skill in the art, and include, for example, saline. Alternatively, such compositions may include conventional delivery systems into which protein of the invention is incorporated. Optionally, these compositions may contain other active ingredients.
The therapeutic agents of this invention may be administered by any appropriate internal route, and may be repeated as needed, e.g., as frequently as one to three times daily for between 1 day to about three weeks to once per week or once biweekly. Preferably, the antagonist antibody is administered less frequently than is the ligand, when it is used therapeutically. The dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient.
As used herein, the term xe2x80x9cpharmaceuticalxe2x80x9d includes veterinary applications of the invention. The term xe2x80x9ctherapeutically effective amountxe2x80x9d refers to that amount of a receptor antagonist antibody, which is useful for alleviating a selected condition. These therapeutic compositions of the invention may be administered to mimic the effect of the normal receptor ligand.
The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host. The altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously or intranasally.
Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the engineered (e.g., humanized) antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier. In the compositions of the invention, an aqueous suspension or solution containing the engineered antibody, preferably buffered at physiological pH, in a form ready for injection is preferred. The compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an engineered antibody of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer""s solution, and about 1 mg to about 30 mg and preferably 5 mg to about 25 mg of an engineered antibody of the invention. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, xe2x80x9cRemington""s Pharmaceutical Sciencexe2x80x9d, 15th ed., Mack Publishing Company, Easton, Pa.
It is preferred that the therapeutic agent of the invention, when in a pharmaceutical preparation, be present in unit dose forms. The appropriate therapeutically effective dose can be determined readily by those of skill in the art. To effectively treat anemia in a human or other animal, one dose of approximately 0.01 mg to approximately 20 mg per kg body weight of a protein or an antibody of this invention should be administered parenterally, preferably i.v. or i.m. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician during the response period.
The present invention will now be described with reference to the following specific, non-limiting examples.